Vibrio cholerae O1 causes the fatal epidemic disease cholera. The ability of V. cholerae to cause disease in humans is dependent upon two primary virulence factors, the toxin-coregulated pilus (TCP), a critical colonization factor, and cholera toxin (CT). The expression of these factors is controlled by a highly regulated transcriptional cascade that serves as a paradigm for the regulation of bacterial virulence. Expression of the cascade is initiated at the tcpPH promoter by a cooperative interaction between the regulators AphA and AphB. TcpPH and ToxRS are homologous pairs of transmembrane regulators that then cooperate to activate expression from the toxT promoter. ToxT, an AraC-type regulator, directly activates the expression of TCP and CT. Transcriptional activation of the virulence cascade is strongly dependent upon a variety of stimuli from the external environment. The long-term goals of this proposal are to understand the molecular basis of virulence gene regulation in V. cholerae so as to facilitate the development of new strategies to control its infectivity. Through a collaborative effort involving laboratories with expertise in structural biology, virulence gene regulation and pathogenesis, we have discovered that exogenous unsaturated fatty acids (UFAs) are capable of binding to ToxT and impairing its ability to activate virulence gene expression. UFAs bind into a ligand pocket in the N-terminal domain of ToxT and inhibit its dimerization as well as its ability to bind to DNA. In the El Tor biotype, the current pandemic strain, we have recently discovered that exogenous UFAs also influence the transcription, translation and stability of ToxT by different mechanisms through the master regulator of FA metabolism, FadR. We have also recently discovered that the virulence regulator ToxR is influenced by a signalling process referred to as regulated intramembrane proteolysis (RIP) similar to that controlling the levels of TcpP. The RIP of ToxR occurs in response to nutrient limitation at alkaline pH, a condition associated with the late stages of infection, which might signal to the bacterium to terminate virulence and prepare for entry back into the environment. This proposal will build upon the ToxT structural and functional data, as well as our recent studies, in order to elucidate several key mechanisms involved in regulating the expression of the virulence cascade. In Aim 1, we propose to determine the mechanisms controlling the dimerization of ToxT and its inhibition by UFAs as well as the small molecule virstatin. In Aim 2, we propose to elucidate how FadR influences both the translation of ToxT and its stability. In Aim 3, we propose to determine the factors involved in the RIP of ToxR and its role in V. cholerae pathogenesis. These studies will contribute significantly toward our understanding of how virulence gene expression is regulated in V. cholerae and will likely provide new avenues for antivirulence drug discovery.